The natural isoflavones, present in most of all vascular plants, constitute a subclass of flavonoids, characterized by presence of two benzene rings linked to a group of three carbon atoms in their linear or ring form.
The main flavone ingredients of seeds Glycine max Merill (soya-bean) constitute β-D-glycosides such as genistin, daidzin and glycitin, while the corresponding to them aglycones (genistein, daidzein and glycitein) occur in up to one hundred times smaller quantities and appear in more considerable amounts only when being technologically processed, under heat treatment or due to fermentation.
Genistein (4′,5,7-trihydroxy-3-phenylchromen-4-on) is a competitive inhibitor for protein tyrosine kinases (PTK), playing important role as a structural analogue of adenosine triphosphate (ATP) (T. J. O'Dell et al., Nature, 353, p.558 (1991). Researches concerning the biological role of the enzymes from the PTK group in the transmission of chemical signals cascade from the cell membrane receptors to the nuclear effectors modulating the gene expression and transcription, constitute at present one of the most promising trends in the medical chemistry (P. W. Groundwater et al., Progr. Med. Chem., 33, p. 233 (1996), at the same time it is expected that the selective phosphorylation inhibitors will give rise not only to a new generation of drugs, for instance antineoplastic drugs, but also to such compounds, which will enable to inhibit the oncogenesis, thus preventing neoplastic diseases.
In medical respect, the genistein is at present classified as phytoestrogen and included among the new class of biological active compounds termed selective estrogen receptors modulators (SERM).
In the Polish patent application nr 346955 and in the publication of K. Polkowski et al. (Cancer Letters 203(2004), 59-69) has been proved the cytotoxic and cytostatic activity in vitro of several ethereal and ester derivatives of genistein, in which one hydrogen atom of at least one hydroxyl group at the positions 7 and/or 4′ has been replaced by fatty acid radical, alkyloaryl or saccharide groups. In this application like as in the Polish patent application nr 354794 have been also shown the manners for functionalizing the hydroxyl groups of genistein and a synthetic methodology applied to obtain new derivatives.
At the base of present invention lies the finding that genistein, like other isoflavones and semisynthetic derivatives thereof, causes a significant inhibition of glycosaminoglycans synthesis and in consequence of it would be useful for treatment of diseases caused by excessive production or storage of mucopolysaccharides.
The mucopolysaccharidoses (MPS) are the rare genetic conditions which inheritance is autosomal recessive (with exception of a mucopolysaccharidose of type II, MPS II, the inheritance of which is X-linked) (Kaye, Curr. Treat Opinions Neurol. 3 (2000), 249). The cause of each of the type of mucopolysaccharidose is a damage of a specific lysosomal enzyme taking part in the degradation of the mucopolysaccharides.
The mucopolysaccharides, at present called glycosaminoglycans (GAG), are chemical compounds produced by the most of tissues in mammals. They are among others responsible for the correct structure and functioning of connective tissue, for proper communication between the cells (including intracellular signaling owing to aided binding of signaling proteins with their receptors in the cell membranes) and for possibility of proper penetration of different substances into body tissues. Most of the glycosaminoglycans occur in form of peptidoglycans, that is to say are connected by a covalent bond (usually by a residue of serine) with a proper peptide. In the regular cell occurs the permanent turnover of the glycosaminoglycans, it means synthesis of the new and degradation of the elder molecules. The breakdown of these compounds in the cells take place in the lysosomes by participation of a dozen or so enzymes specifically directed to these organelles (Kaplan et al., Proc. Natl. Acad. Sci. USA 74(1977), 2026).
In case that one of the enzymes responsible for the breakdown of the mucopolysaccharides is deficient or its activity significantly decreased, they will not be degraded and accumulate in the lysosomes and in the intercellular space. The insufficiency of the lysosomal apparatus stimulates many compensatory processes, after depletion of which the complicated function and structure of the cell will be disturbed, leading to its destruction and in consequence of it gives rise to characteristic clinic symptoms. In the pathomechanism of these diseases the key significance has not only the mechanic results of the storage, but also the toxic and damaging effect of the accumulated compounds and cytokines.
Different types of mucopolysaccharidose disorders classified as Type I through IX and the deficient enzymes are listed in Table 1.
TABLE 1Mucopolysaccharidoses classification*TypeName of syndromeEnzyme deficientMPS I-HHurler syndromeα-L-iduronidaseMPS I-SScheie syndromeα-L-iduronidaseMPS I-H/SHurler-Scheieα-L-iduronidasesyndromeMPS IIHunter syndromeiduronate sulphataseMPS III ASanfilippo syndromeheparan-N-sulphatasetype AMPS III BSanfilippo syndromeN-acetyl-α-D-glucosaminidasetype BMPS III CSanfilippo syndromeCoA-α-glucosaminide-N-type CacetyltransferaseMPS III DSanfilippo syndromeN-acetyl-α-D-glucosaminide-6-type DsulfataseMPS IV AMorquio syndromeN-acetyl-α-D-glucosaminide-6-type AsulfataseMPS IV BMorquio syndromeB-galactosidasetype BMPS VIMaroteaux-LamyN-acetylgalactosamine-4-syndromesulfatase (acetylsulfatase)MPS VIISly syndromeB-glucuronidaseMPS IX—hyaluronidase*Neufeld, E. F. and Muenzer, J., The mucopolysaccharidoses. In: Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D. (ed.): The metabolic and molecular bases of inherited diseases. New York: McGraw-Hill Co, 2001, 3421-3452;Wegrzyn G., Mukopolisacharydozy. Praktyka i Klinika Medyczna, 4/5 (2000), 5-18.
The accumulation of mucopolysaccharides in the lyzosomes causes the gradually function impairment of cells, tissues and practically all the organs. These diseases have a progressing character and a middle time of patient's survival amounts a dozen or so years.
Up to the present time in case of all mucopolysaccharidoses only symptomatic treatment was possible, not very efficient, although it could to a certain degree improve the comfort of life of the sick people. Some hopes were set on bone marrow transplantation, in order to introduce the cells producing the lacking enzymes to the organism of the sick person (Schiffmann and Brady, Drugs, 62(2002), 733). This method proved not to be very efficient and at the same time it is connected with a higher risk of complications. Since recently replacement therapy of mucopolysaccharidose of type I became possible, based on intravenous administration of the lacking recombinant enzyme—the α-L-iduronidase (Kakkis, Expert Opin. Investig. Drugs, 11(2002), 675). Although clinical research has shown a very high efficacy of this type of treatment towards most of the organs, it must be say that because of the blood-brain barrier as a serious problem remain disturbances in functioning of the central nervous system, found in part of patients with MPS 1 (especially in type MPS 1-H).
Enzymatic replacement therapy in case of MPS 1 is actually the only one accessible method of causal treatment of mucopolysaccharidoses. Other MPS types are not treated at all or only symptomatic treatment could be applied, which proved to be not very effective. Consequently there is an urgent necessity to look for therapeutic methods for this group of chronic diseases, which in the absence of treatment lead to the premature death of patients.
The greatest problem by introducing enzymatic replacement therapy in other MPS types is the fact that in many types of this disease (MPS II, MPS IIIA, MPS IIIB, MPS IIIC, MPS IIID, MPS VII) severe neurological symptoms may occur related to the central nervous system, while in others types the largest changes are observed in the osteoarticular system (MPS IVA, MPS IVB, MPS VI). The penetration of the intravenously administered enzyme into the central nervous system is minimal, whereas into the bone is very impeded.
An approach to the treatment of mucopolysaccharidoses being alternative to the enzymatic replacement therapy can constitute the inhibition of the synthesis of the substrate, which can not be degraded in the organisms of sick individuals (Wegrzyn et al., Med. Hypothes., 62 (2004), 986).
The present invention is based on an unexpected finding that the natural isoflavone, genistein, added to the cultured fibroblasts derived from patients affected with MPS, in the concentration range of 10-30 micromoles/l, causes significant inhibition of glycosaminoglycans synthesis. The incubations of cells derived from patients affected with different MPS types (MPS I, MPS II, MPS IIIA and MPS IIIB) with genistein has proved that in these cells the level of glycosaminoglycans not only did not increase but on the contrary significantly decreased, reaching after six days a level almost identical with the normal one. The results of these tests have been confirmed by electron microscope investigations of the cells, where the disappearance of deposits in fibroblasts cultured during one week in presence of genistein (in concentration of 10 micromolar units) has been observed. Similar effects were observed in consequence of fibroblasts incubation in presence of a soya-bean isoflavones extract, what indicates that these compounds and their derivatives could have similar activity as in the case of genistein.
The molecular mechanism of genistein activity as inhibitor of glycosaminoglycans synthesis has not been recognized, although it seems to be likely that it is related to the previous founding to inhibit tyrosine kinase activity of the epidermal growth factor receptor (EGFr) (Akiyama et al., J. Biol. Chem., 262(1987), 5592). This factor is in turn essential for the effective glycosaminoglycans synthesis (Tirone et al., J. Biol. Chem., 272(1997), 4787). Activation of EFGr can probably stimulate the system of intracellular signal transmission, leading to the effective expression of genes, which are coding the enzymes related to the process of glycosaminoglycans synthesis. The fact, already proved, that the intravenously administered genistein crosses the blood-brain barrier in a rat with an effectiveness of about 10% (Tai, J. Chromatogr. A 1073(2005), 317), opens further possibilities for treating some neurological symptoms in patients suffering from mucopolysaccharidoses, which at present is entirely impossible.